MicroRNAs.
MicroRNAs (miRNAs) are a class of short (˜18-24 nucleotides) noncoding RNAs that regulate gene expression at the post-transcriptional level2. Depending on the degree of homology to their target sequences, miRNA binding induces either translational repression or cleavage of target mRNAs2. As powerful gene regulators, miRNAs play important roles in development, cell differentiation, and regulation of cell cycle, apoptosis and signaling pathways2,3. Aberrant expression of miRNAs has been found in all types of tumors4,5; the different cancer types have distinct miRNA expression profiles6. Specific miRNAs including some miRNA families containing a few nucleotide differences are constantly released from the primary tumor into blood stream and present in an incredible stable form7. Recent studies demonstrated that circulating miRNAs are enveloped inside exosomal vesicles and can be transferable and functional in the recipient cells8,9. Thus, detection of tumor specific circulating miRNAs provides a powerful tool for early diagnosis, staging, and monitoring of cancer cells10.
MiRNA Detection.
Several technologies including reverse transcription real-time PCR (RT-qPCR) and microarray for miRNA detection have been developed11-13. Each technology has its own advantages, but limitations include requiring enzymatic amplification and semi-quantitative results14. In particular the short miRNA sequences make it difficult to selectively design the primers or probes, resulting in cross-hybridization and low selectivity. This is especially true when the miRNAs contain a few or a single nucleotide difference in a miRNA family. Emerging techniques based on colorimetry, bioluminescence, enzyme turnovers and electrochemistry have been proposed, and nanoparticles and molecular beacon have been applied to miRNA detection with high sensitivity and selectivity (review14). But the intrinsic versatility needs to be improved. Recently, the integration of single-molecule fluorescence and lock-nucleic acids (LNA)15 probes provided a sensitive method for miRNA profiling in tissue samples16, though this method requires expensive instrument.
Nanopore Single Molecule Detection.
In a nanometer-scaled pore structure, the ion current becomes very sensitive to the presence, location and conformation of single target molecules occupying the ion pathway17. This sensitivity allows “visualizing” single molecules, elucidating their kinetics from characteristic change in the pore conductance, and quantifying the target from the occurrence of single molecule signature events. Nanopores have been developed as receptive single molecule detectors for broad biotechnological applications (reviews17-19). The nanopore is also recognized as one of the next generations of DNA sequencing technologies20,21. For example, the 2-nm nanopore, α-hemolysin transmembrane protein pore, allows rapid translocation of single-stranded oligonucleotide, which has been well characterized for DNA sequencing22-27. However, the molecular translocation-based sensing mode is not suitable for miRNA detection because the sequences of all mature miRNAs are short (18-24 nt), and when traversing the nanopore, the current signals by different miRNAs are indistinguishable.
Therefore, there is a need to provide a new miRNA detection method based on nano-scale pore structure with improved sensitivity, speedy process, and cost efficiency.